Die Interessenabnahme von Schülerinnen und Schülern in der Sekundarstufe 1 in allen naturwissenschaftlichen Fächern ist nicht nur bekannt, es liegen bereits diverse Untersuchungen über die Ursachen dieser Abnahme vor. Dabei standen sowohl die fachlichen Inhalte als auch die Tätigkeiten im Unterricht im Fokus der Untersuchungen. Warum aber gerade in der Oberschule das Interesse abnimmt und warum eine derartig starke Abnahme des Interesses nicht bereits in der Grundschule zu beobachten ist, bleibt zu eruieren. Ein direkter Vergleich zwischen der Interessenentwicklung von Schülerinnen und Schülern von der 5. zur 6. Jahrgangsstufe l in Berliner Oberschulen und Grundschulen im Fach Naturwissenschaften soll zur Klärung dieser Frage beitragen. Bei gleichen fachlichen Inhalten kann der Fokus ganz auf den Unterricht und dessen Umsetzung gelegt werden. In der vorliegenden Arbeit wird untersucht, ob Grundschullehrkräfte in ihrem Unterricht das Interesse der Schülerinnen und Schüler besser fördern als Oberschullehrkräfte. Dahingehend wird eine Querschnittstudie sowohl an Grund- als auch an Oberschulen mit Schülerinnen und Schülern der 5. und 6. Jahrgangsstufe durchgeführt. Erhoben werden das Interesse und die Motivation der Lernenden am Unterricht und die im Unterricht eingesetzten Unterrichtsmethoden. Als Basis für eine Förderung der Interessenentwicklung wird die Befriedigung der psychologischen Grundbedürfnisse (Deci & Ryan, 1993) zugrunde gelegt. Die Ergebnisse der Untersuchung weisen keine nennenswerten Unterschiede zwischen den Unterrichts-methoden von Grundschul- und Oberschullehrkräften auf. Gleichzeitig zeigt sich eine Interessenabnahme über die Klassenstufen an Ober- und Grundschulen. Des Weiteren wird ein eindeutiger Unterschied in der Befriedigung der psychologischen Grundbedürfnisse nach Kompetenzerleben, Autonomieerleben und sozia-ler Eingebundenheit über die Schulformen hinweg deutlich. Die Unterschiede in der Befriedigung der psy-chologischen Grundbedürfnisse lassen den Schluss zu, dass es dennoch einen Unterschied im Unterricht zwischen Grund- und Oberschulen geben muss. Darüber hinaus geben Sie Anlass zu der Vermutung, dass der Schulwechsel als solcher einen entscheidenden Einfluss auf die Interessenentwicklung hat. / The decline of student interest in the natural sciences, which occurs in secondary school (7th – 10th) grades, is not only a known fact, but also has been the subject of many research studies to discover its causes. In these studies, both subject matter and classtime activities were the primary focus. Why precise-ly this interest declines in the upper grades and why such a marked decline in interest is not already ob-servable in primary school are the questions at hand. A direct comparison of the development of student interest in the natural sciences between the 5th and 6th grades in a regular primary school as well as those in a special secondary school (grundständiges Gym-nasium), which begins with the 5th grade, can contribute to the clarification of these questions. If the sub-ject matter is the same, the emphasis can be on the instruction itself and its implementation. This thesis presents a study that elucidates if primary school teachers are more capable of stimulating the interests of students in their classes than their special secondary school counterparts. For that reason a cross-sectional study of students in the 5th and 6th grades at primary schools and special secondary schools was conducted in which the interest and motivations of students and the teaching methods em-ployed were examined. In this study, the satisfaction of psychological basic needs served as the basis for the stimulation of inter-est development (Deci & Ryan, 1993). The results of this research show no substantial differences between the teaching methods of primary and special secondary school teachers. However, the decline of interest between the grades examined could be verified across primary and special secondary schools. Furthermore, a clear difference in the satisfaction of psychological basic needs across school forms could be shown. These differences suggest, that there has to be a distinction between educa-tion in primary and special secondary schools after all. In addition one can conclude from this research that the progression to a higher school level itself has a decisive impact on interest development.

Objetos de aprendizagem (OA) compõem uma das abordagens adotadas para tratar a crescente complexidade da educação com auxílio de computadores, promovendo o reúso e a qualidade de materiais didáticos. Tais benefícios podem ser ampliados ao incluir questões de projeto instrucional e associá-las às características multimídias e interativas dos dispositivos computacionais, sistematizando-se a produção de objetos de aprendizagem. Nesse contexto, esta tese define uma abordagem para o desenvolvimento de objetos de aprendizagem multimídias e interativos considerando o cenário de televisão digital. Essa abordagem, denominada LOD (Learning Object Development) consiste de um modelo de processo ou processo-padrão para desenvolvimento de objetos de aprendizagem (SPLOD Standard Process for Learning Object Development), a partir do qual se define um processo (LODP Learning Object Development Process) adequado para a construção de objetos de aprendizagem como recursos educacionais abertos (análogos a software livre); de um método de desenvolvimento dirigido a modelos, denominado LODM (Learning Object Development Method), compreendendo a modelagem conceitual, instrucional e de interação do objeto de aprendizagem; e de um conjunto de ferramentas que estabelecem um protótipo de ambiente para o desenvolvimento de objetos de aprendizagem (LODE Learning Object Development Environment) considerando o método LODM e o processo LODP. A abordagem LOD foi preliminarmente avaliada quanto à engenharia de objetos de aprendizagem multimídia e interativos, representados por apresentações multimídia e aplicações interativas para televisão digital para a plataforma Ginga do Sistema Brasileiro de Televisão Digital (SBTVD). Os resultados apontam para os benefícios da abordagem integrada para o desenvolvimento, com a geração de objetos de aprendizagem multimídias e interativos de forma mais ágil e sistemática, além de promover o reúso desde os primeiros passos da modelagem dos objetos de aprendizagem / Learning objects are an approach to address the increasingly complexity of computersupported education, fostering reuse and learning material quality. Such benefits can be improved by considering instructional design and the multimedia and interactivity features of current computing devices, providing a systematic method to develop learning objects. Considering such context, this thesis defines an approach to the development of multimedia and interactive learning objects, considering the digital television scenario. This approach is named LOD (Learning Object Development) and is composed of (1) a reference process (SPLOD Standard Process for Learning Object Development) from which we defined a process for learning objects development (LODP Learning Object Development Process) suitable for engineering of open educational resources; (2) a model-driven development method (LODM Learning Object Development Method) that integrates conceptual, instructional and interaction modeling of learning objects; and (3) a set of tools that establish a prototype for an environment for learning object development named LODE (Learning Object Development Environment). The LOD approach was evaluated with multimedia and interactive learning objects development realized as multimedia presentations and interactive digital television applications aimed at the Brazilian Digital Television System and its Ginga middleware. The results evince the benefits of the integrated approach, providing an agile and systematic development and fostering the reuse since the initial models of the learning object engineering process

Magister Public Health - MPH (Public Health) / The presence of highly effective medicines has made very little impact in reducing deaths as a result of tuberculosis (TB), a curable condition but when managed inappropriately, may result in Drug Resistant TB. TB accounts for about one in four deaths that occur in HIV positive people and HIV has been found to be a risk factor for complex unfavorable outcomes in MDR TB patients and a very strong predictor for death and default. The relationship between diabetes and TB has also been explored, with some authors identifying diabetes as a risk factor for TB, and with related poor clinical outcomes in both conditions when they co-exist. Exploring the clinical characteristics and treatment outcomes of MDR TB patients in the presence of these risk factors could present an opportunity to provide better care through increased case-detection activities, improved clinical management and better access to care for all these conditions. The aim of the study was to describe the clinical characteristics and treatment outcomes of MDR TB patients initiated on treatment at Nkqubela and Fort Grey Hospitals.

In this thesis, we study local Tb theorems for singular integral operators in the setting of spaces of homogeneous type. We give a direct proof of the local Tb theorem with L^2 integrability on the pseudo- accretive system. Our argument relies on the Beylkin-Coifman-Rokhlin algorithm applied in adapted Haar wavelet basis and some stopping time results. Motivated by questions of S. Hofmann, we extend it to the case when the integrability conditions are lower than 2, with an additional weak boundedness type hypothesis, which incorporates some Hardy type inequalities. We study the possibility of relaxing the support conditions on the pseudo-accretive system to a slight enlargement of the dyadic cubes. We also give a result in the case when, for practical reasons, hypotheses on the pseudo-accretive system are made on balls rather than dyadic cubes. Finally we study the particular case of perfect dyadic operators for which the proof gets much simpler. Our argument gives us the opportunity to study Hardy type inequalities. The latter are well known in the Euclidean setting, but seem to have been overlooked in spaces of homogeneous type. We prove that they hold without restriction in the dyadic setting. In the more general case of a ball B and its corona 2B\B, they can be obtained from some geometric conditions relative to the distribution of points in the homogeneous space. For example, we prove that some relative layer decay property suffices. We also prove that this property is implied by the monotone geodesic property of Tessera. Finally, we give some explicit examples and counterexamples in the complex plane to illustrate the relationship between the geometry of the homogeneous space and the validity of the Hardy type inequalities.

The purpose of this study was to explore factors related to poor adherence to TB treatment in Keetmanshoop municipal area in Namibia. A qualitative, exploratory design was used to explore basic information about the study. Participants were using purposive sampling technique. The researcher-implemented triangulation, based on three different categories of participants, namely Field Promoters (preferred). DOT supporters (more preferred), and TB patients (most preferred). Participants were chosen in a ratio of preference. 1:2:3 respectively. The transcripts and audio tapes from interviews were analysed using the thematic content analysis. Five main themes emerged. In order of descending prominence, these themes included: factors leading to lack of adherence to TB treatment, support to enhance adherence to treatment, existing behaviours determining adherence, suggestions to promote adherence to treatment and practices to promote adherence to treatment. / Health Studies / M.A. (Public Health)

In this thesis, a mathematical model for HIV and TB co-infection with TB treatment among populations of Ethiopia is developed and analyzed. The TB model includes an age of infection. We compute the basic reproduction numbers RTB and RH for TB and HIV respectively, and the overall repro- duction number R for the system. We find that if R < 1 and R > 1; then the disease-free and the endemic equilibria are locally asymptotically stable, respectively. Otherwise these equilibria are unstable. The TB-only endemic equilibrium is locally asymptotically stable if RTB > 1, and RH < 1. How- ever, the symmetric condition, RTB < 1 and RH > 1, does not necessarily guarantee the stability of the HIV-only equilibrium, but it is possible that TB can coexist with HIV when RH > 1: As a result, we assess the impact of TB treatment on the prevalence of TB and HIV co-infection. To derive and formulate the nonlinear differential equations models for HIV and TB co-infection that accounts for treatment, we formulate and analyze the HIV only sub models, the TB-only sub models and the full models of HIV and TB combined. The TB-only sub model includes both ODEs and PDEs in order to describe the variable infectiousness and e ect of TB treatment during the infectious period. To analyse and solve the three models, we construct robust methods, namely the numerical nonstandard definite difference methods (NSFDMs). Moreover, we improve the order of convergence of these methods in their applications to solve the model of HIV and TB co-infection with TB treatment at the population level in Ethiopia. The methods developed in this thesis work and show convergence, especially for individuals with small tolerance either to the disease free or the endemic equilibria for first order mixed ODE and PDE as we observed in our models. / Mathematical Sciences / Ph. D. (Applied Mathematics)

Objetos de aprendizagem (OA) compõem uma das abordagens adotadas para tratar a crescente complexidade da educação com auxílio de computadores, promovendo o reúso e a qualidade de materiais didáticos. Tais benefícios podem ser ampliados ao incluir questões de projeto instrucional e associá-las às características multimídias e interativas dos dispositivos computacionais, sistematizando-se a produção de objetos de aprendizagem. Nesse contexto, esta tese define uma abordagem para o desenvolvimento de objetos de aprendizagem multimídias e interativos considerando o cenário de televisão digital. Essa abordagem, denominada LOD (Learning Object Development) consiste de um modelo de processo ou processo-padrão para desenvolvimento de objetos de aprendizagem (SPLOD Standard Process for Learning Object Development), a partir do qual se define um processo (LODP Learning Object Development Process) adequado para a construção de objetos de aprendizagem como recursos educacionais abertos (análogos a software livre); de um método de desenvolvimento dirigido a modelos, denominado LODM (Learning Object Development Method), compreendendo a modelagem conceitual, instrucional e de interação do objeto de aprendizagem; e de um conjunto de ferramentas que estabelecem um protótipo de ambiente para o desenvolvimento de objetos de aprendizagem (LODE Learning Object Development Environment) considerando o método LODM e o processo LODP. A abordagem LOD foi preliminarmente avaliada quanto à engenharia de objetos de aprendizagem multimídia e interativos, representados por apresentações multimídia e aplicações interativas para televisão digital para a plataforma Ginga do Sistema Brasileiro de Televisão Digital (SBTVD). Os resultados apontam para os benefícios da abordagem integrada para o desenvolvimento, com a geração de objetos de aprendizagem multimídias e interativos de forma mais ágil e sistemática, além de promover o reúso desde os primeiros passos da modelagem dos objetos de aprendizagem / Learning objects are an approach to address the increasingly complexity of computersupported education, fostering reuse and learning material quality. Such benefits can be improved by considering instructional design and the multimedia and interactivity features of current computing devices, providing a systematic method to develop learning objects. Considering such context, this thesis defines an approach to the development of multimedia and interactive learning objects, considering the digital television scenario. This approach is named LOD (Learning Object Development) and is composed of (1) a reference process (SPLOD Standard Process for Learning Object Development) from which we defined a process for learning objects development (LODP Learning Object Development Process) suitable for engineering of open educational resources; (2) a model-driven development method (LODM Learning Object Development Method) that integrates conceptual, instructional and interaction modeling of learning objects; and (3) a set of tools that establish a prototype for an environment for learning object development named LODE (Learning Object Development Environment). The LOD approach was evaluated with multimedia and interactive learning objects development realized as multimedia presentations and interactive digital television applications aimed at the Brazilian Digital Television System and its Ginga middleware. The results evince the benefits of the integrated approach, providing an agile and systematic development and fostering the reuse since the initial models of the learning object engineering process

Magister Public Health - MPH / Background: Although clinicians encounter antimycobacterial treatment in Human mmunodeficiency (HIV)-infected children as one of the most common treatments coadministered with antiretroviral treatment (ART), quantitative data on the extent of antimycobacterial treatment among HIV-infected children at the time of commencement of ART and at different times during ART is scarce. The baseline risk factors associated with being on both ART and antimycobacterial treatments are not known and it remains to be elucidated how the different exposure factors impact on the antimycobacterial treatment-free survival of children who begin ART without antimycobacterial treatment.Objectives: To describe the prevalence of antimycobacterial treatment among children at the time of starting ART and the antimycobacterial treatment-free survival after starting ART. Design: A retrospective cohort study based on record reviews at the Harriet Shezi children&lsquo;s clinic (HSCC).Population: HIV-infected children less than fifteen years of age presumed ART naïve started on ART at HSCC.Analysis: A descriptive analysis of the prevalence of antimycobacterial treatment at time of start of ART was done. Kaplan Meier (KM) survival curves were used to determine the antimycobacterial treatment-free survival and logistic regression was used to analyze the association between baseline factors and future antimycobacterial treatment among children who had no antimycobacterial treatment at time of start of ART. Results: The prevalence of antimycobacterial treatment at the time of starting ART was 518/1941 (26.7%, 95% confidence interval (CI): 24.7-28.7). Among children who started ART without antimycobacterial treatment, the KM cumulative probability of antiretroviral and antimycobacterial (ART/antimycobacterial) co-treatment in the first 3 months of starting ART was 4.6% (95% CI: 4.1- 5.2), in the first 12 months it was 18.1% (95% CI: 17.0-19.2) and in the first 24 months of starting ART it was 24% (95% CI: 21.9-25.1). Survival analysis suggested that children with high baseline viral load, advanced World Health Organization (WHO) stage of disease, very low normalized weight for age (waz) and very young age (less than one year) at start of ART had significantly reduced antimycobacterial treatment-free survival (log rank p < 0.05) in the first two years of starting ART. In the logistic regression model, age less than one year {Odds ratio (OR): 3.7 (95% CI: 2.2-6.0; p <0.0001)} and very low weight for age Z-score (waz < -3) {OR; 2.2 (95% CI: 1.4-3.6; p = 0.0015)} were the two critical risk factors independently associated with future antimycobacterial treatment. Conclusions: Antimycobacterial treatment is extremely common among HIV-infected children at the time of starting ART and early after starting ART and the incremental risk of being on ART/antimycobacterial co-treatment decreases with time on ART. The results emphasize the need for a heightened and careful alertness for mycobacterial events especially among children starting ART with severe malnutrition and those who start ART at age less than one year. The results further suggest that it is probably optimal to start ART in children before their nutritional status has deteriorated severely in the course of the HIV disease so that they get protection against mycobacterial events by early ART. / South Africa

Magister Public Health - MPH / Background: Tuberculosis (TB) poses a major public health challenge in South Africa and in the Northern Cape Province. The province has the third highest in TB incidence rate in the country. Poor adherence to TB treatment impacts negatively on treatment outcomes. Siyanda district in the Northern Cape Province has the second highest number of TB defaulters in the province despite the fact that 79.9% of these patients are on Community Based Direct Observation of Treatment (CBDOT). Aim: To explore the reasons for defaulting of TB patients from TB treatment in the CBDOT Programme in the Siyanda district, Northern Cape Province Study design: This was a qualitative exploratory study. Study population and sampling: Two TB nurses with varying years of experience in the TB Control Programme serving as key informants were selected from the participating facilities in the study area. Ten TB defaulters who were on the CBDOT programme were selected from the Electronic TB Register. Two focus group discussions (FGDs) comprising of purposively selected DOT Supporters (five in one group and six in the other) from different NGOs in the community were selected for maximum variation. Data collection: Key informant interviews were conducted with the TB nurses. Records of all defaulters in the study population were reviewed including clinic progress notes and patient TB treatment cards. In-depth interviews were conducted with the TB patients. FGDs were conducted with DOT supporters. Analysis: Analysis commenced simultaneously with collection of data. This enabled the researcher to continuously review and reflect on the data collected. Thematic content analysis was done.Categories emerged through the inductive process of the data analysis. Notes that were kept during data collection, reflections, audiotapes and transcripts were used to support the thick description of the findings. Results: The participants generally appreciated the programme and mostly had a good relationship with the DOT supporters. However, the quality of care exacerbated by inadequate health services such as lack of adherence counselling training of health professionals, low levels of education amongst TB defaulters, were found to be major contributory factors to TB defaulting. The patients interrupted treatment several times before defaulting, were not counselled during the interruption phase and understood TB messages differently. TB defaulters in the Siyanda District face socio economic challenges which include alcohol abuse, a major historic ill in the district and the grape farming community in the region. The impact of the disability grant on TB treatment adherence remains anecdotal and requires further research as TB defaulters did not admit to defaulting so that they could continue benefiting from the disability grant although these statements were refuted by the DOT supporters and key informants. The attitude of employers and fear of losing employment were also contributing factors. Conclusion and recommendations: It has become evident that TB in the Siyanda District is a public health issue. The predominantly rural, impoverished and transient community that moves to the farms to seek employment requires a CBDOT programme that will address pertinent challenges in the district to achieve a positive reduction in the TB defaulter rate. It will require collaboration with stakeholders including farmers, to address the challenges posed by the disease. Improved staff allocation, staff capacity development and community education are also recommended to improve quality of care.

The current, grim world-TB scenario, with TB being the single largest infectious disease killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003). This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited. The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development. In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase. Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded. The availability of only three crystal structures for a critical enzyme like dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter. It is interesting to note that the P-loop-containing nucleoside monophosphate kinases (NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches. As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom. Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.